Zanthoxylum bungeanum tissue culture medium and application thereof

Abstract

The present invention provides a Zanthoxylum bungeanum tissue culture medium and its application. It belongs to the technical field of plant tissue culture. By optimizing the culture medium system, a bud induction culture medium and a rooting culture medium for Dahongpao Zanthoxylum bungeanum tissue culture are constructed, integrating the callus tissue culture, adventitious bud induction culture, and adventitious bud proliferation culture processes in the prior art into one culture process. In this process, the bud induction culture medium of the present invention can effectively induce the formation of clustered buds, greatly simplifying the cultivation process; using Dahongpao petioles as explants provides new material support for the rapid propagation of Zanthoxylum bungeanum tissue culture; using the culture medium and tissue culture method of the present invention to tissue culture Dahongpao Zanthoxylum bungeanum petioles, the adventitious bud induction rate is 93.33%, the proliferation coefficient is 7.4, and the rooting rate is 90%, which is extremely efficient.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, in particular to a Zanthoxylum bungeanum tissue culture medium and application thereof. Background Art

[0002] Zanthoxylum bungeanum is a widely cultivated, important woody oil-bearing and spice tree species in my country. It is also one of the key nationally developed advantageous specialty economic forests and natural plant spice species. It possesses economic, medicinal, and ecological value, and boasts broad development prospects. In recent years, the cultivation of Dahongpao Zanthoxylum bungeanum has grown rapidly, becoming a pillar of specialty agricultural products in many regions. It boasts the largest and most extensive planting area in Zanthoxylum bungeanum production. Research on the tissue culture system of Dahongpao Zanthoxylum bungeanum is of great significance for the establishment of an industrialized asexual propagation system for Dahongpao Zanthoxylum bungeanum, the selection and breeding of high-quality Dahongpao Zanthoxylum bungeanum varieties, and the further development and utilization of plant resources.

[0003] Currently, the commonly used tissue culture methods for Zanthoxylum plants mainly include four cultivation processes: callus culture, adventitious bud induction culture, adventitious bud proliferation culture, and rooting culture. The cultivation process is cumbersome, and the explants are usually young leaves or stem segments with buds. The explant selection is limited, and production efficiency is low. For example, Xu Xu, "Research on the Tissue Culture Technology System of Hanyuan Dahongpao Zanthoxylum bungeanum," Sichuan Agricultural University (2014), used stem segments with buds as explant materials and carried out Dahongpao Zanthoxylum bungeanum tissue culture through the steps of callus induction culture, proliferation culture, and rooting culture. However, the induction rate, rooting rate, and growth coefficient were 72.8%, 66.7%, and 4.6, respectively, which were not ideal, and the process was relatively cumbersome. Qin Sujie et al., "In vitro culture of compound leaves and high-frequency plant regeneration of Japanese pepper," Economic Forest Research (2018), used young leaves of Japanese pepper as explant materials. The entire tissue culture process only used two culture media, but the growth coefficient and rooting rate were 5.42% and 83%, respectively, which were also less than ideal. However, there is no technical solution for Dahongpao Zanthoxylum bungeanum with simple process and good tissue culture effect in the prior art.

[0004] Therefore, how to change the current situation in the prior art where the explant selectivity of Dahongpao Zanthoxylum bungeanum tissue culture is single, the culture process is cumbersome, and the rooting induction rate is low is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a Zanthoxylum bungeanum tissue culture medium and an application thereof. By optimizing the culture medium system, a bud induction culture medium and a rooting culture medium for Dahongpao Zanthoxylum bungeanum tissue culture are constructed, and the callus tissue culture, adventitious bud induction culture, and adventitious bud proliferation culture processes in the prior art are integrated into one culture process. The bud induction culture medium of the present invention can effectively induce the formation of clustered buds, greatly simplifying the cultivation process; Dahongpao petioles are used as explants, providing new material support for the rapid propagation of Zanthoxylum bungeanum tissue culture; and the tissue culture medium and method of the present invention are used to tissue culture Dahongpao Zanthoxylum bungeanum, with an adventitious bud induction rate of 93.33%, a proliferation coefficient of 7.4, and a rooting rate of 90%, which is extremely efficient.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A Zanthoxylum bungeanum tissue culture medium comprises a bud induction medium and a rooting medium; the bud induction medium comprises WPM, 0.1-1.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, and 0.01-0.05 mg / L TDZ; and the rooting medium comprises WPM, 0.5-1.5 mg / L IBA, and 0.01-0.5 mg / L NAA.

[0008] The beneficial effects of the above operation are: compared with the traditional Zanthoxylum bungeanum tissue culture medium composed of callus tissue culture medium, adventitious bud induction culture medium, adventitious bud proliferation culture medium and rooting culture medium, the culture medium of the present invention only includes bud induction culture medium and rooting culture medium, which effectively simplifies the culture process, and the culture medium of the present invention is better than the traditional culture medium in promoting induction, rooting and survival.

[0009] Another object of the present invention is to provide an application of the Zanthoxylum bungeanum tissue culture medium in Dahongpao Zanthoxylum bungeanum tissue culture.

[0010] Preferably, the Zanthoxylum bungeanum tissue is petiole tissue.

[0011] The beneficial effect of the above operation is that the petiole tissue of Dahongpao Zanthoxylum bungeanum has a stronger regeneration ability than tender leaves and stem segments with buds as explants, which helps to obtain a higher induction rate, rooting rate and survival rate during tissue culture.

[0012] Another object of the present invention is to provide a Zanthoxylum bungeanum tissue culture method, using the above-mentioned Zanthoxylum bungeanum tissue culture medium, specifically as follows:

[0013] (1) Preparation of explants: petioles of Dahongpao Zanthoxylum bungeanum were selected and sterilized to obtain explants;

[0014] (2) Bud induction culture: The explants from step (1) are inoculated into a bud induction medium for induction culture. The bud induction medium is replaced every 2-3 weeks. Buds emerge after 10 days of culture. After 30-40 days of culture, a large number of clustered buds are obtained.

[0015] (3) Rooting culture: The clustered buds induced in step (2) are inoculated into a rooting medium and cultured for 40 to 50 days to obtain rooted seedlings;

[0016] (4) Transplantation: Before transplanting, place the rooted seedlings together with the tissue culture bottles outdoors for 5 to 7 days. Then take out the seedlings, clean the root culture medium and transplant them into the culture medium. Put the tissue culture bottle upside down on the seedlings and culture them for 15 to 20 days to obtain potted seedlings.

[0017] Preferably, the aseptic treatment process in step (1) is as follows: the petioles of the current year leaves of Dahongpao are placed in saturated detergent water for washing, and then rinsed in running water for 2-3 hours, and then placed in 75% alcohol for 1 minute and 0.1% mercuric chloride for 5-7 minutes.

[0018] Preferably, the culture conditions in step (2) and step (3) are as follows: the culture temperature is 25±2°C, the photoperiod is: light / darkness = 16h / 8h, and the light intensity is 2000-3000 lux.

[0019] Preferably, before use, the bud induction medium in step (2) and the rooting medium in step (3) need to be added with 3.5-4.5 g agar and 20-30 g sucrose per liter of culture medium, and the culture medium pH is 5.8-6.0.

[0020] Preferably, the culture medium in step (4) is as follows: vermiculite: peat soil = 3:1.

[0021] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The explants of the present invention are the petiole tissues of young leaves of Dahongpao Zanthoxylum bungeanum, which have stronger regeneration ability than traditional young leaves and stem segments with buds. They are crucial for establishing a stable and efficient plant regeneration system and improving the induction rate and rooting rate. This is the first time in the Zanthoxylum bungeanum tissue culture system.

[0023] (2) The present invention integrates the three processes of callus induction, adventitious bud induction, and proliferation culture into one culture process. The same culture medium - bud induction culture medium can be used to effectively induce the formation of clustered buds. While ensuring a high induction rate, growth coefficient, and rooting rate, the culture process is greatly simplified.

[0024] (3) The culture medium and culture method of the present invention were used to carry out tissue culture on Dahongpao Zanthoxylum bungeanum, with an adventitious bud induction rate of 93.33%, a proliferation coefficient of 7.4, and a rooting rate of 90%, which are extremely efficient.

[0025] (4) The bud induction medium of the present invention is also suitable for tissue culture of stem segments with buds. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0027] Figure 1 The attached figure shows the sprouting of petiole buds after 15 days of induction culture;

[0028] Figure 2 The attached figure shows the sprouting of petiole buds after 30 days of induction culture;

[0029] Figure 3 The attached figure shows the budding of petiole buds after 60 days of induction culture;

[0030] Figure 4 The attached figure shows a complete plant after 40 days of cultivation of clustered shoots and rooting;

[0031] Figure 5 The attached figure shows the rooting situation of clustered buds after 40 days of rooting culture. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Example 1

[0034] A Zanthoxylum bungeanum tissue culture medium comprises a bud induction medium and a rooting medium; the bud induction medium comprises WPM, 0.1-1.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, and 0.01-0.05 mg / L TDZ; and the rooting medium comprises WPM, 0.5-1.5 mg / L IBA, and 0.01-0.5 mg / L NAA.

[0035] Example 2

[0036] A Zanthoxylum bungeanum tissue culture method is as follows:

[0037] (1) Explant preparation: 1 cm of petiole tissue from young leaves of Dahongpao Zanthoxylum bungeanum growing in the current year was collected in April or May. The surface dust was gently brushed off with a brush in saturated detergent water. After washing, the tissue was placed in running water for 2 hours. The tissue was then soaked in 75% alcohol for 1 minute, soaked in 0.1% mercuric chloride for 5 minutes, and washed twice with sterile water to obtain the explant.

[0038] (2) Bud induction culture: Sterile petioles were inoculated into bud induction medium (WPM + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.03 mg / L TDZ). 3.5-4.5 g agar and 20-30 g sucrose were added per liter of medium. The induction culture was carried out at a temperature of 25 ± 2 °C, a photoperiod of 16 h / 8 h (light / dark), a light intensity of 2000 lux, and a pH of 6.0. The bud induction medium was replaced every 2 weeks. A large number of clustered buds were obtained after 30 days of culture.

[0039] (3) Rooting culture: The clustered buds induced in step (2) were inoculated into a rooting medium (WPM + 0.5 mg / LIBA + 0.05 mg / L NAA), 3.5-4.5 g agar and 20-30 g sucrose were added per liter of culture medium, and rooting culture was carried out under the conditions of a temperature of 25 ± 2°C, a photoperiod of (light / dark) 16 h / 8 h, a light intensity of 2000 lux, and a pH of 5.8. Rooted seedlings were obtained after 40 days of culture.

[0040] (4) Transplantation: Before transplanting, place the rooted seedlings together with the tissue culture bottles outdoors for 5 days. Then take out the seedlings and wash off the culture medium attached to the roots under running water. Transplant them into a matrix with a ratio of vermiculite to peat soil of 3:1. Water them thoroughly and place the tissue culture bottles on the seedlings. After 15 days, remove the tissue culture bottles to obtain potted seedlings.

[0041] Example 3

[0042] A Zanthoxylum bungeanum tissue culture method is as follows:

[0043] (1) Explant preparation: 2 cm of petiole tissue from young leaves of Dahongpao Zanthoxylum bungeanum growing in the current year was collected in April or May. The surface dust was gently brushed off with a brush in saturated detergent water. After washing, the tissue was placed in running water for 3 hours, then soaked in 75% alcohol for 1 minute, soaked in 0.1% mercuric chloride for 7 minutes, and washed three times with sterile water to obtain the explant.

[0044] (2) Bud induction culture: Sterile petioles were inoculated into bud induction medium (WPM + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.05 mg / L TDZ). 3.5-4.5 g agar and 20-30 g sucrose were added per liter of medium. The induction culture was carried out at a temperature of 25 ± 2 °C, a photoperiod of 16 h / 8 h (light / dark), a light intensity of 3000 lux, and a pH of 6.0. The bud induction medium was replaced every 2 weeks. After 35 days of culture, a large number of clustered buds were obtained.

[0045] (3) Rooting culture: The clustered buds induced in step (2) were inoculated into a rooting medium (WPM + 1.5 mg / LIBA + 0.01 mg / L NAA), 3.5-4.5 g agar and 20-30 g sucrose were added per liter of culture medium, and rooting culture was carried out under the conditions of a temperature of 25 ± 2°C, a photoperiod of (light / dark) 16 h / 8 h, a light intensity of 3000 lux, and a pH of 6.0. Rooted seedlings were obtained after 33 days of culture.

[0046] (4) Transplantation: Before transplanting, place the rooted seedlings together with the tissue culture bottles outdoors for 5 days. Then take out the seedlings and wash off the culture medium attached to the roots under running water. Transplant them into a matrix with a ratio of vermiculite to peat soil of 3:1. Water them thoroughly and place the tissue culture bottles on the seedlings. After they continue to grow for 17 days, potted seedlings are obtained with a survival rate of over 90%.

[0047] Example 4

[0048] The adventitious bud induction rate, growth coefficient, rooting rate and survival rate of the bud induction culture process of the culture method of the present invention were statistically analyzed. At the same time, control experiments with different materials and culture medium compositions were conducted. The experimental results are shown in Table 1:

[0049] Table 1: Statistics of adventitious bud induction rate, growth coefficient, rooting rate and survival rate

[0050]

[0051] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of a Zanthoxylum bungeanum tissue culture medium in Dahongpao Zanthoxylum bungeanum tissue culture, characterized in that: The Zanthoxylum bungeanum tissue culture medium comprises a bud induction medium and a rooting medium; the bud induction medium is WPM+0.5-1.0 mg / L 6-BA+0.1-0.2 mg / L NAA+0.03-0.05 mg / LTDZ; the rooting medium is WPM+0.5-1.5 mg / L 1BA+0.01-0.05 mg / L NAA.

2. The use according to claim 1, characterized in that The Zanthoxylum bungeanum tissue is petiole tissue.

3. A method for Zanthoxylum bungeanum tissue culture, characterized in that: The Zanthoxylum bungeanum tissue culture medium according to claim 1 is used, specifically as follows: (1) Preparation of explants: petioles of Dahongpao Zanthoxylum bungeanum were selected and sterilized to obtain explants; (2) Bud induction culture: The explants from step (1) are inoculated into a bud induction medium for induction culture. The bud induction medium is replaced every 2-3 weeks. Buds emerge after 10 days of culture. After 30-40 days of culture, a large number of clustered buds are obtained. (3) Rooting culture: The clustered buds induced in step (2) are inoculated into a rooting medium and cultured for 40-50 days to obtain rooted seedlings; (4) Transplantation: Before transplanting, place the rooted seedlings together with the tissue culture bottles outdoors for 5-7 days. Then take out the seedlings, clean the root culture medium, transplant them to the culture medium, turn the tissue culture bottle upside down on the seedlings, and culture them for 15-20 days to obtain potted seedlings.

4. the Zanthoxylum bungeanum tissue culture method according to claim 3, is characterized in that, The aseptic treatment process of step (1) is as follows: the petioles of the current year leaves of Dahongpao are placed in saturated detergent water for washing, and then placed in running water for 2-3 hours, and then placed in 75% alcohol for 1 minute and 0.1% mercuric chloride for 5-7 minutes.

5. the Zanthoxylum bungeanum tissue culture method according to claim 3, is characterized in that, The culture conditions in step (2) and step (3) are as follows: the culture temperature is 25±2°C, the photoperiod is: light / darkness=16h / 8h, and the light intensity is 2000-3000lux.

6. The Zanthoxylum bungeanum tissue culture method according to claim 3, wherein Before use, the bud induction medium in step (2) and the rooting medium in step (3) need to be added with 3.5-4.5 g of agar and 20-30 g of sucrose per liter of culture medium, and the culture medium pH is 5.8-6.

0.

7. The Zanthoxylum bungeanum tissue culture method according to claim 4, wherein The culture medium in step (4) is as follows: vermiculite: peat soil = 3:1.

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